DESCRIPTION: This proposal is in response to the Funding Opportunity Announcement RFA-RM-13-021 Exceptionally Innovative Tools and Technologies for Single Cell Analysis (R21). Our proposal addresses the goals stated in the Research Objectives: to develop next-generation tools that distinguish heterogeneous states among cells in situ, in particular Tools that provide significant advances in sensitivity, selectivity or spatiotemporal resolution of molecules/structures/activities within single cells and between ostensibly similar cells in situ. Gene activation patterns vary widely in complexity. Some are straightforward and static, such as the pattern of many housekeeping genes, that are expressed in all cells at all times in life. Others are extraordinarily intricate with expression fluctuating from minute to minute or from cell to cell. Changes in gene expression reflect changes in the transcriptional landscape of the cell, which in many cases occur in response to extracellular signals. Characterizing the spatiotemporal changes in gene expression is therefore a critical question in cell and developmental biology. In addition, besides temporal dynamics within particular cells, the spatial architecture of cell-cell connection and communication are also critical. In multicellular organisms, direct intercellular contacts play vital roles in almost every biological process, from basic functions like cell proliferation, differentiation, and migration; to higher functions like immune system activation, body movement, and memory formation. To address some of the limitations with current technologies, we propose to develop a series of tools that will provide significant advances in the analysis of cellular heterogeneity and cell contact in vivo. Specifically, we propose to develop: Aim 1. Sensitive sensors of gene expression to visualize heterogeneity among groups of cells within tissues. Transcriptional reporters that drive the expression of fluorescent proteins (FPs) are commonly visualized to analyze the activation of pathways; however, the stability of these reporters makes it impossible to achieve the temporal resolution needed to dissect dynamic gene expression changes. We have begun to develop a series of new fluorescence reporters (Dynamic Fluorescence Reporters) that improve dramatically the temporal visualization of gene activities to address these fundamental questions. We propose to further optimize these tools and demonstrate their in vivo applications to: 1. study heterogeneity among apparently homogeneous cell populations; and 2. examine the transcriptional response of individual cells to a morphogen molecule. Aim 2. Transcriptional sensors to detect contacts between cells. We have developed robust in vitro synthetic ligand-receptor systems that activate a transcriptional reporter or effector following direct cell-cell contact. We propose to establish these systems in vivo and evaluate their use for: 1. the study of cytonemes, cellular projections making long-range contact with other cells, in imaginal disc epithelia; and 2. the study of neuronal connectivity. The tools developed in Aims 1 and 2 will have many additional applications and enable studies of cross-talk between signaling pathways; blood cell homing; the binding of secreted ligands to their cellular targets by generating secreted ligand-GFP molecules; as well as isolation of specific cell types at different developmental/functional states for RNAseq transcriptome analyses. Finally, although the experiments described in this R21 will be performed in Drosophila, the tools once established will be easily transferrable to other systems, especially vertebrates.